Generation and characterization of hybridoma antibodies for immunotherapy of tularemia.

Department of Pathology and Laboratory Medicine, Boston University School of Medicine, Boston, MA 02118, USA.

Abstract

Tularemia is caused by the Gram-negative facultative intracellular bacterium Francisella tularensis, which has been classified as a category A select agent-a likely bioweapon. The high virulence of F. tularensis and the threat of engineered antibiotic resistant variants warrant the development of new therapies to combat this disease. We have characterized 14 anti-Francisella hybridoma antibodies derived from mice infected with F. tularensis live vaccine strain (LVS) for potential use as immunotherapy of tularemia. All 14 antibodies cross-reacted with virulent F. tularensis type A clinical isolates, 8 bound to a purified preparation of LVS LPS, and 6 bound to five protein antigens, identified by proteome microarray analysis. An IgG2a antibody, reactive with the LPS preparation, conferred full protection when administered either systemically or intranasally to BALB/c mice post challenge with a lethal dose of intranasal LVS; three other antibodies prolonged survival. These anti-Francisella hybridoma antibodies could be converted to chimeric versions with mouse V regions and human C regions to serve as components of a recombinant polyclonal antibody for clinical testing as immunotherapy of tularemia. The current study is the first to employ proteome microarrays to identify the target antigens of anti-Francisella monoclonal antibodies and the first to demonstrate the systemic and intranasal efficacy of monoclonal antibodies for post-exposure treatment of respiratory tularemia.

Derivation of anti-LVS hybridoma antibodies. Antibodies are grouped based on the mouse strain of origin (BALB/cJ or C57BL/6J), the immunization protocol and day of fusion, and the isotype (determined with an isotyping ELISA kit). Also listed are the non-LVS binding hybridoma antibody 19, used as negative control and IgM isotype control; the commercially available hybridoma antibody FB11, specific for the O-antigen polysaccharide chain of F. tularensis LPS, used as positive control; and the anti-human colorectal cancer hybridoma antibody CO17-1A, used as IgG isotype control. Antibodies further characterized in the current study are bolded.

Reactivity of hybridoma antibodies with virulent F. tularensis type A clinical isolates and a purified LVS LPS preparation. Hybridoma supernatants containing 1 and 0.04 μg/ml of antibody were compared by ELISA for binding to LVS, heat-killed LVS, a mixture of eight heat-killed F. tularensis type A clinical isolates (Left), or for binding to LVS, equal OD of E. coli TG1 (or equal CFU which yielded the same results) and LPS. The names (numbers) of the antibodies are indicated. FB11, the positive control, is indicated by “+” and antibodies 19 and CO17-1A, the negative controls, are indicated by “−”. OD values that exceeded the capacity of the spectrophotometer (2.5) were recorded as 2.5. Background values (of wells that received no hybridoma supernatants, generally 0.04–0.05 OD) were subtracted from all other OD values.

Immunoblot analysis of LPS-binding hybridoma antibodies. LVS lysate (A) or purified LPS preparation (B) was subjected to SDS-PAGE under reducing conditions and transferred to a nitrocellulose membrane. The membrane was cut into strips and each strip probed with the indicated hybridoma antibody at 2.5 μg/ml for Ab2–6, and 0.5 μg/ml for FB11 and Ab7–9. FB11, specific for the O-antigen polysaccharide chain of F. tularensis LPS was used as standard. The most clearly visible of several similar immunoblots are shown, and the estimated positions of molecular weight markers (in kDa) are indicated.

Proteinase K (PK) sensitivity of target antigens of anti-LVS hybridoma antibodies. One half of a sample of LVS lysate was treated with PK, then each the untreated and PK-treated samples were subjected to SDS-PAGE under reducing conditions and transferred to nitrocellulose membranes. The membranes were cut into strips and each strip probed with the indicated hybridoma antibody. Ab7, an LPS-binder, was used as negative control for PK-treatment. The untreated (A) and PK-treated (B) membrane strips for each antibody were developed simultaneously and for the same time. Immunoblots from three separate experiments are shown; the nitrocellulose membranes in the middle and right immunoblots were “aged” for two weeks prior to use to allow visualization of the bands identified by antibodies 10 and 17. The positions of molecular weight markers (in kDa) are indicated for the left immunoblot.

Identification of the target antigens of hybridoma antibodies by proteome microarray analysis. Separate pads of Chip 1 were probed with the indicated IgG or IgM hybridoma antibodies at antibody concentrations ranging from 9 to 141 μg/ml, followed by isotype-specific secondary antibodies. Antibody 17, at a concentration of 150 μg/ml, was re-tested on Chip 2. Microarray spots giving the highest fluorescence intensity signals on each pad are circled and the FTT numbers of the corresponding proteins are indicated. The fluorescence intensities of the circled spots are indicated for antibody 17.

Changes in body weight (BW) of mice inoculated i.n with LVS. Groups of 10 five-week-old BALB/cJ mice were exposed to the indicated number of LVS CFU and weighed daily. The daily BW of each mouse was divided by its initial (pre-LVS exposure) BW and the mean BW ratio (in %) ± SD was plotted for each group as a function of time.